Sustained transduction of ocular cells with a bovine immunodeficiency viral vector.

Human immunodeficiency viral (HIV) vectors mediate long-term transduction of many types of nondividing cells in vivo. Bovine immunodeficiency virus (BIV) is a lentivirus that shares many characteristics with HIV, but does not cause human disease. In this study, we investigated the potential of BIV vectors for ocular gene therapy. An enhanced green fluorescent protein (eGFP)-encoding reporter gene was packaged in recombinant BIV vector (BIV.eGFP). Adult C57BL/6 mice were given an intravitreous (5 x 10(4) or 5 x 10(5) transducing units [TU]) or subretinal (5 x 10(5) TU) injection of BIV.eGFP and then GFP expression was assessed at several time points. In vivo examinations of mice showed that subretinal injection of BIV.eGFP resulted in strong expression of GFP from the first examination at 1 week through the final examination at 20 weeks. Only a few mice that received intravitreous injection of BIV.eGFP showed GFP expression by ocular examinations until 11-12 weeks, when most showed small areas of expression. Postmortem examinations showed prominent GFP expression in retinal pigmented epithelial (RPE) cells throughout the region of subretinal injection of vector, although occasional negatively staining RPE cells were scattered among the much more numerous, brilliantly staining cells. Ciliary epithelial cells frequently expressed GFP, as did occasional Müller cells and rarely other retinal cells. The expression was stable from the first time point (2 weeks) to the last (20 weeks). Postmortem examination of eyes given an intravitreous injection of BIV.eGFP showed transduction of cells in the corneal endothelium and a few scattered retinal cells. There was no evidence of inflammation or toxicity in any eyes. These data show that BIV vectors mediate rapid and sustained transduction of RPE cells, suggesting that they may be useful for ocular gene therapy targeting RPE cells.

Cibenzoline plasma concentration and antiarrhythmic effect.

The relationship between plasma concentrations of cibenzoline and its antiarrhythmic effect was evaluated in patients receiving the drug orally as part of an ascending multiple dose efficacy and tolerance study. Twenty-five patients participated in a 3-day placebo period, 3 days of 32.5 mg cibenzoline every 6 hr, 3 days of 65 mg cibenzoline every 6 hr, 3 days of 81.25 mg cibenzoline every 6 hr, and 3 final placebo days. Arrhythmia frequency was monitored by 24-hr Holter monitoring and blood samples were drawn during and after dosing. Percent reduction in baseline premature ventricular complex (PVC) frequency for the 25 subjects demonstrated considerable interpatient variability in antiarrhythmic response. Cibenzoline plasma concentrations over 300 ng/ml were associated with some decrease in PVC frequency in virtually all cases. The relationship between plasma concentration and PVC frequency was studied more rigorously in eight of the 25 patients and that for ventricular couplet (VC) frequency was studied in six. For these analyses, PVC and VC frequency data were averaged over 6-hr intervals and plotted against trough cibenzoline concentrations. The data from each patient were fitted with a concentration-effect function (Hill equation) by means of least squares regression. With the exception of two extreme values, the concentration corresponding to 90% reduction in PVC frequency (C90) ranged from 215 to 405 ng/ml. In five of the six patients with arrhythmia in whom VC data were also evaluated, the individual C90 for VCs were considerably less than those for PVCs. The agreement between the observed concentration-response relationships and those predicted by curve-fitting the data suggests that the antiarrhythmic effect of cibenzoline is proportional to its plasma concentration, and that the Hill equation provides an accurate mathematic description of the concentration-response relationship.

Age and cibenzoline disposition.

Oral cibenzoline kinetics were followed in 36 healthy subjects aged from 22 to 78 yr divided into groups of six subjects per decade between 20 and 80 yr. Each received a single, oral, 160-mg dose of cibenzoline. Blood and urine samples were collected for 72 hr. Cibenzoline plasma and urine concentrations were measured by HPLC. Maximum plasma cibenzoline concentrations (Cmax) ranged from 283 to 1100 ng/ml and occurred 1 to 2.5 hr after dosing. Apparent oral clearance (ClT) ranged from 401 to 1677 ml/min and the t 1/2 ranged from 5.9 to 13.4 hr. Nonrenal clearance (ClNR) ranged from 65 to 1113 ml/min, renal clearance (ClR) ranged from 165 to 645 ml/min, and 31% to 86% of the dose was recovered unchanged in urine (Xu). The volume of distribution (Vd) was large, ranging from 236 to 948 l. There was a significant relationship between age and the following kinetic parameters: Cmax, Xu, t 1/2 (all of which increased with age), ClT, ClR, ClNR, the terminal elimination rate constant beta, and Vd (which decreased with age). Mean ClT was 999 +/- 371 ml/min in the 20- to 30-yr age group and was 465 +/- 78 ml/min in the 70- to 80-yr age group. The change in ClT with age resulted from a decreased in both ClR and ClNR. Mean t 1/2 varied from 7 hr in the youngest group to 10.5 hr in the oldest group. The age-related changes in cibenzoline kinetics occurred over the entire age range studied and the relationship between age and these kinetic parameters appeared to be linear.(ABSTRACT TRUNCATED AT 250 WORDS)

Human recombinant epidermal growth factor in experimental corneal wound healing.

Human recombinant epidermal growth factor (hEGF) was evaluated in various corneal wound healing models in the rabbit. Human EGF accelerated epithelial wound healing in corneal reepithelialization, anterior-keratectomy, and alkali-burn models at concentrations of 10-500 micrograms/ml given four times daily (qid). In the corneal reepithelialization model, 100 micrograms/ml of hEGF qid produced a 45% increase in the wound-healing rate compared with control (0.13 versus 0.09 mm/hr) with a similar response at 500 micrograms/ml qid. In the anterior-keratectomy model, 500 micrograms/ml of hEGF qid accelerated healing by 40% (0.07 versus 0.05 mm/hr), although the 100 micrograms/ml dose was not active in this model, and 1 microgram/ml of hEGF actually slowed the healing rate. In the alkali-burn model, 10 and 100 micrograms/ml of hEGF qid for 32 days appeared to produce faster initial healing of the wound compared with control, although the wound recurred in both hEGF and control groups. These results suggest that hEGF may be helpful in some epithelial disorders in humans, although considerations of dose response and optimal dosing regimens must be addressed.

Periocular injection of microspheres containing PKC412 inhibits choroidal neovascularization in a porcine model.

PURPOSE: Oral administration of PKC412, a kinase inhibitor that blocks several isoforms of protein kinase C (PKC) and receptors for vascular endothelial growth factor (VEGF), platelet-derived growth factor, and stem cell factor, inhibits ocular neovascularization in a murine model. The purpose of this study was to determine whether sustained local delivery of PKC412 in a human-sized eye inhibits choroidal neovascularization (CNV). METHODS: Laser photocoagulation was used to rupture Bruch's membrane in young domestic pigs, and then a periocular injection of control microspheres or microspheres containing 25% or 50% PKC412 was given. After 10 days the integrated area of CNV at Bruch's membrane rupture sites was measured by image analysis. The levels of PKC412 in choroid, retina, and vitreous were measured either 10 or 20 days after periocular injection of 50% PKC microspheres or at 20 days after injection of 25% PKC412 microspheres. RESULTS: The areas of CNV at Bruch's membrane rupture sites were significantly smaller in eyes that received a periocular injection of microspheres containing 25% (P=0.0042) or 50% (P=0.0012) PKC412 than those in eyes injected with control microspheres. Ten days after periocular injection of 50% PKC412 microspheres, PKC412 was detected in the choroid, but not in the retina or vitreous. Twenty days after periocular injection of 50% PKC412, high levels of PKC412 were measured in the choroid, vitreous, and retina. Levels were lower but still substantial in all three compartments 20 days after periocular injection of 25% microspheres. CONCLUSIONS: Sustained local delivery of PKC412 provides a promising approach for treatment of CNV.

Pharmacokinetics of oral cibenzoline in arrhythmia patients.

The pharmacokinetics of oral cibenzoline were studied in 30 arrhythmia patients as part of an ascending multiple-dose efficacy study. The elimination half-life of the drug following repetitive dosing ranged from 7.6 to 22.3 hours, with a harmonic mean of 12.3 hours (n = 24), and increased with age and decreasing renal function. The drug exhibited apparent dose proportional and linear pharmacokinetics over the range of doses studied. Multivariate analysis revealed that the patients' age and serum creatinine concentration accounted for 71% of the variability in the range of beta values (terminal elimination rate constant), and that 69.5% of the intersubject variability in the steady-state trough plasma concentrations could be accounted for by the patients' age, weight and serum creatinine concentration. These data suggest that, although there is some intersubject variability in the elimination and accumulation of cibenzoline, much of the variability can be explained by the patients' age, weight and renal function.

Controversy I: Patients or healthy volunteers for pharmacokinetic studies?

Many factors should be considered when choosing an appropriate population for a pharmacokinetic trial. Although there are some generalities that apply to most studies, each investigation must be judged separately, since the relevant considerations will vary depending on the particular study, the nature of the drug, and the population that will receive it for therapeutic benefit. Some of the most important information that is generated from pharmacokinetic studies concerns the pharmacokinetic variability among patients and the factors that can influence this variability under the conditions that the drug will be used. This information can best be obtained from a combination of baseline studies to define the variability within the patient population(s) and comparative studies to determine the impact of specific variables on the disposition of the drug and its pharmacokinetic variability. These data can provide valuable information to the clinician that can be used to individualize drug dosage and optimize therapy as well as to identify populations who may be at high risk of therapeutic failure or drug toxicity. It is our feeling that baseline studies in patients are necessary for understanding the pharmacokinetics of a drug, whereas the objectives of most comparative studies can be achieved using healthy volunteers. For most comparative studies, the data obtained from healthy volunteers will reflect what will occur in patients, especially if the variable of interest is drug absorption. This is particularly important when practical and ethical considerations preclude the use of patients. When considering studies in the elderly, one must decide whether the variable of interest may be influenced by age.(ABSTRACT TRUNCATED AT 250 WORDS)

Verapamil pharmacodynamics after intravenous and oral dosing: theoretic consideration.

Differences in the potency of intravenous (IV) and oral verapamil have recently been reported with the concentration-response curve for PR-interval prolongation being shifted further to the right following oral administration relative to IV administration. Using well-established pharmacokinetic models, a theoretic basis for these observations is presented. Simultaneous curve fitting of the IV and oral verapamil plasma concentration and PR-interval data to a single pharmacokinetic-pharmacodynamic model allowed prediction of differences in the pharmacodynamic potency of verapamil as a function of the rate of drug administration. These data indicate that the rate of input of drug into the systemic circulation can influence the rate and extent of entry of drug into an effect compartment, which in turn can result in different plasma concentration-response relationships.

Pharmacokinetic and pharmacodynamic modeling of cibenzoline plasma concentrations and antiarrhythmic effect.

The plasma concentration and antiarrhythmic effect data following multiple, ascending oral doses of cibenzoline in four patients with frequent premature ventricular contractions (PVCs) were analyzed using pharmacokinetic and pharmacodynamic modeling. Three methods of data analysis were tested in the analysis of the large amount of arrhythmia frequency data gathered during the study: as total-data set, average-data set, and grouped-data set. We have shown that the antiarrhythmic effect profile of the drug could be characterized by average data when a large number of PVC measurements are involved. Using the average-data sets, the plasma concentration of the drug at steady state could be correlated to the antiarrhythmic response using pharmacokinetic and pharmacodynamic modeling.

Red blood cell sorbitol lowering effects and tolerance of single doses of AL 1576 (HOE 843) in diabetic patients.

The safety and biochemical effects of AL 1576 (HOE 483), a recently developed aldose reductase inhibitor, were evaluated. In a double-blind, placebo-controlled, clinical trial, AL 1576 (HOE 483) was administered to diabetic patients for the first time. Four single, orally administered dose levels were tested, (2, 5, 10, and 20 mg). No clinically important adverse effects were seen in any of the patients. AL 1576 (HOE 483) suppressed red blood cell (RBC) sorbitol concentrations in a dose-related fashion. Also found were statistically significant inverse correlations between the plasma drug concentration and both RBC sorbitol concentrations as well as RBC sorbitol/serum glucose ratios. In single doses up to 20 mg, AL 1576 (HOE 483) is well tolerated and decreases RBC sorbitol, a biochemical marker of pharmacologic activity, in diabetic patients.

Pharmacokinetics and pharmacodynamics of intravenous cibenzoline in normal volunteers.

The pharmacokinetics of intravenous (IV) cibenzoline were studied in six healthy male volunteers ranging in age from 51 to 78 years. The subjects received intravenous (IV) cibenzoline 100 mg over 20 minutes, and plasma and urine specimens were collected for 48 hours. Cibenzoline plasma concentrations at the end of the infusion ranged from 730 to 1,420 ng/mL and exhibited triexponential decline thereafter. The following mean model independent pharmacokinetic parameters were calculated from the plasma and urine concentration data: terminal half-life, 9.8 hours (range, 8.5-11.9); plasma clearance, 523 mL/min (range, 387-687); volume of distribution, 445 L (range, 328-506); and renal clearance, 289 mL/min (range, 202-334). Approximately 31% to 59% of the dose was recovered unchanged in the urine in 48 hours. A triexponential pharmacokinetic equation with zero order input was used to curve fit the plasma and urine data, and the model-dependent parameters agreed well with the model-independent estimates. A hysteresis loop was observed in the relationship between cibenzoline plasma concentration and QRS prolongation, indicating an initial lag between plasma concentration and effect after IV administration. Based on these results, the following preliminary dosing regimen was proposed to rapidly achieve and maintain therapeutic plasma concentrations equal to or slightly greater than 200-400 ng/mL: 0.25 mg/kg/min IV bolus over one minute followed by 1-1.5 mg/kg/hr for one hour and 0.2-0.4 mg/kg/hr for long-term infusion.

Single-dose pharmacokinetics and dose proportionality of oral cibenzoline.

The pharmacokinetics of cibenzoline were evaluated in four young healthy volunteers who received ascending oral doses of 65, 97.5, 130, 162.5, 195, 227.5, and 260 mg separated by one week. Cibenzoline plasma concentrations exhibited an apparent biexponential decline following oral absorption. Maximum plasma concentrations and area under the plasma concentration-time curve increased in proportion to the dose. The mean elimination half-life among subjects was independent of dose and ranged from 7.3 to 8.7 hours. Oral clearance ranged from 380 to 575 ml/min and was also independent of dose. A single pharmacokinetic equation was used to adequately describe the plasma concentration data over the entire range of doses for each subject, indicating dose-proportional and linear pharmacokinetics.

The influence of liver dysfunction on the pharmacokinetics of carprofen.

The pharmacokinetics of the investigational agent carprofen were examined in 12 patients with liver dysfunction (hepatic cirrhosis) and in six normal volunteers following single 100-mg oral administration of carprofen. In addition, three patients with acute hepatitis received a single 100-mg dose during the acute phase of the disease, and two of these patients received the same dose after they had convalesced. The pharmacokinetic parameters and urinary excretion data did not differ significantly (P greater than 0.05) between patients with hepatic cirrhosis and healthy volunteers. The mean +/- SD area under plasma concentration-time curve and apparent oral plasma clearance values were 57.8 +/- 11.7 micrograms X h/mL and 30.0 +/- 6.3 mL/min, respectively, in patients and 52.4 +/- 11.3 micrograms X h/mL and 33.1 +/- 7.2 mL/min in normals. The respective harmonic mean elimination half-lives were 10.5 and 9.4 hours. The 0-24 hour urinary recovery of intact drug and the glucuronide conjugate were 7.0 +/- 4.9% and 28.9 +/- 11.0%, respectively, in patients compared to 5.5 +/- 7.1% and 20.1 +/- 12.3% in normal subjects. The results of this study showed that liver dysfunction (hepatic cirrhosis) had no effect on the pharmacokinetic profile of carprofen. In the two patients with acute hepatitis who completed the study, the results suggest that the apparent oral clearance of carprofen may increase during the acute phase of the disease.

Isolated perfused rabbit lung as a model for intravascular and intrabronchial administration of bronchodilator drugs II:Isoproterenol prodrugs.

The pulmonary disposition of two diester prodrugs of isoproterenol (di-p-toluoylisoproterenol and dipivaloylisoproterenol) was studied in the isolated perfused rabbit lung preparation. High-pressure liquid chromatographic methods were developed to measure diester, monoester, isoproterenol, and 3-O-methylisoproterenol from a single 1-ml perfusate sample. The prodrugs were administered directly into the circulating perfusion medium and by endotracheal instillation. Perfusate concentrations of diester, monoester, isoproterenol, and 3-O-methylisoproterenol were measured for 180 min. The diesters were rapidly eliminated from the perfusate with a subsequent increase in monoester concentrations. Isoproterenol levels were observed within minutes of prodrug administration, peaked at 60-80 min, and declined slowly thereafter. The prodrugs were rapidly absorbed following endotracheal administration with 30-50% of the diester being metabolized during the first pass through the lung.

Isolated perfused rabbit lung as a model for intravascular and intrabronchial administration of bronchodilator drugs I:Isoproterenol.

The absorption, uptake, and metabolism of isoproterenol was studied following intravascular, intrabronchial, and aerosol administration of the drug to the isolated perfused rabbit lung. Capacity-limited metabolism of isoproterenol was observed following the addition of five doses, ranging from 10(-7) to 10(-5) moles, directly into the circulation of the lung system. A physiologically based perfusion model was developed to describe the disposition of the drug and metabolite in the isolated lung preparation. This model was also used to analyze data collected following intrabronchial and aerosol administration of isoproterenol.

High-performance liquid chromatographic assay of the aldose reductase inhibitor spiro-(2-fluoro-9H-fluorene-9,4'-imidazolidine)-2',5'-dione (AL01567) in plasma and urine and its pharmacokinetics in humans.

Two selective high-performance liquid chromatographic (HPLC) methods have been developed for the quantitative determination of spiro-(2-fluoro-9H-fluorene-9,4'-imidazolidine)-2',5'-dione (AL01567; 1) in plasma and urine, with an assay sensitivity of 0.25 micrograms/mL for plasma and 0.13 micrograms/mL for urine. The plasma assay procedure involved precipitation of proteins with acetonitrile followed by dilution with water. The diluted supernatant was analyzed on an ODS column eluting with acetonitrile:0.5% phosphoric acid (30:70) adjusted to pH 7.2 with concentrated ammonium hydroxide. The urine assay procedure involved extraction of 1 with 10% n-butanol in hexane, followed by back extraction with 0.05 M sodium hydroxide. The basic extract was neutralized and analyzed on a phenyl column eluting with acetonitrile:10 mM potassium phosphate (30:70; monobasic, pH 5.6). The pharmacokinetics of 1 was investigated in humans following single and multiple oral doses. The elimination half-life from 12 normal subjects following single 100-400-mg oral doses was independent of dose, and the overall mean half-life was 66 +/- 9 h. The overall mean oral clearance (assuming a bioavailability of 100%) was 11 +/- 3 mL/min, and the mean apparent volume of distribution was 59 +/- 13 L. The mean urinary recovery of intact drug during the first 24 h after dosing was 1.2 +/- 0.4% of the administered dose. During once daily 100-mg oral dosing of 1 to five subjects for 21 d, plasma concentrations of 1 reached apparent steady-state by 7 d.(ABSTRACT TRUNCATED AT 250 WORDS)

Disposition of the aldose reductase inhibitor AL01576 in rats.

The disposition of the aldose reductase inhibitor AL01576 was studied in rats following intravenous and oral dosing. Single 4-mg/kg intravenous bolus and oral doses of [14C] AL01576 were administered and levels of radioactivity in blood, excreta, and various tissues were determined over a 168-h period. The decline of radioactivity in blood was quite similar for the two routes of administration, with an apparent half-life of approximately 30 h. At 120 to 144 h, a second, slower elimination phase began that was not fully characterized in the 168-h duration of the study. The HPLC analysis of plasma samples revealed intact AL01576 as the only compound in plasma. The mean plasma parent and radioactivity concentrations are in agreement; suggesting the absence of or an insignificant amount of metabolite in the plasma. The urinary and fecal excretion rate data showed a kinetic pattern similar to that of blood radioactivity. Fecal excretion was the primary route of elimination following both intravenous and oral dosing, accounting for 59% of the administered intravenous dose and 61% of the oral dose. Urinary excretion accounted for 32% of the intravenous dose and 29% of the oral dose. Negligible amounts of radioactivity were recovered as expired 14CO2. Experiments with bile-duct cannulated rats confirmed that the major route of elimination of the drug is biliary excretion. The pattern of distribution of [14C] AL01576 in tissues was quite similar following the two routes of administration. Tissue radioactivity concentration peaked at 4 h (the first sampling time) following both routes of administration in all tissues except the GI tract.(ABSTRACT TRUNCATED AT 250 WORDS)